The present invention relates to an efficient, reliable plant regeneration method using mature seed-derived callus in zoysiagrass (Zoysia japonica Steud.), to the optimised conditions for callus induction and growth from mature seeds, and to the morphological criteria for the selection of callus types that are readily regenerated to mature plants. The invention is critical for plant tissue culture and genetic transformation in this monocot species. Prior to plant regeneration, the cultured callus cells may be genetically transformed with gene expression cassettes.
Turfgrass is widely used for decorative and ground-protective purposes, including golf courses, athletic fields, home gardens, and recreation parks, to improve spatial enjoyment, to decrease dust from ground, and to keep the air clean. As a result, turfgrass is rapidly emerging as a potential commercial target for plant biotechnological applications in recent years. It forms a second largest market size among crop plants in USA, and its application is rapidly growing worldwide. In addition, recent successful achievements of genetic transformation of some turfgrass species have prompted intensive biotechnological researches.
Useful target traits for the genetic manipulation of turfgrass include those to improve ground-covering capacity, tolerance to traffic injury, and regrowth after damage. Resistance to pathogenic infections and adaptability to cold and drought stress are additional targets to be engineered. Among these target traits, the most attractive one is to decrease the cost for regular maintenance, such as watering, mowing, and pathogen control. In USA, more than thirty billion dollars are annually spent just for the regular maintenance. One easy way to achieve these goals is to introduce foreign genes whose physiological activities have been proven in model plants into turfgrass. This method has several advantages over the classical breeding methods. The new trait(s), as a result of such genetic manipulations, can be predicted from the functional analysis in model plants, in which molecular and morphological analysis techniques for the genetic manipulation and tissue culture are well established. It is also possible to introduce more than one gene by a single round of genetic manipulation into any plant species in a predictable way.
However, a technical barrier to be overcome with the turfgrass genetic transformation is that this plant species is extremely reluctant to genetic manipulation, and the tissue culture conditions have not been precisely defined yet, like most of the monocot species. Although a few turfgrass species have been successfully transformed in recent years, the results are not routinely reproducible, and its application to other turfgrass species is very limited so far. This entails that molecular and physiological techniques and systems should be further defined for routine application of the genetic transformation technique to variable turfgrass species. The critical parameters to be refined include the conditions optimized for callus induction and growth and the optimal media compositions, such as nutrients, growth hormones, salt composition, and ionic strength, for plant regeneration from such callus tissues.
The Zoysia japonica Steud., also known as Korean grass, is a species of the warm season turfgrass and widely distributed in the Far-Eastern Asia, including Korea and Japan, and in the temperate zone. Its use is rapidly expanding in USA and other countries in recent years due to its extraordinary characteristics, such as resistance to drought and cold stress and capacity to rapidly recover from traffic damage. It also grows well in poor soil in virtually all climates. Due to these traits, it is widely used for golf courses, athletic fields, roadsides, home gardens, and riverbanks. As its market size is rapidly growing, new varieties with improved resistance to pathogens, herbicides, and environmental stress are demanded by customers. Classical breeding methods have been used to develop such traits, but many laboratories and institutes are striving to find molecular biological methods to genetically engineer the turfgrass. One essential prerequisite for this genetic manipulation is an efficient plant tissue culture and regeneration system. Seed-derived calli and immature embryonic cells have been evaluated for this purpose, and various combinations of media and plant growth hormones have been tested. However, the reported conditions for callus induction and growth and plant regeneration varies among different turfgrass species, and even the calli from a specific species exhibit a broad range of diversity in terms of callus morphology and structural organization, depending on the callus induction conditions.
With rapid accumulation of technological information on the plant tissue culture and genetic transformation in plants, a gene of interest can now be routinely introduced into any desired plants to enhance commercial value, yield, and environmental adaptability. In the present invention, we provide the optimised conditions for callus induction and growth, the morphological clues for isolation of functionally homogeneous callus, and the efficient system for plant regeneration of the zoysiagrass.
As used herein, the term xe2x80x9ccallusxe2x80x9d refers to a group of undifferentiated cells derived from any plant parts of higher plants, preferentially those from mature seeds of the zoysiagrass. The term xe2x80x9cplant regenerationxe2x80x9d refers to the generation of fully differentiated plants from undifferentiated plant tissues, especially callus.
The present invention relates to an experimental method to efficiently induce and grow calli from mature seeds of the zoysiagrass that can be readily used for the genetic transformation. Such an experimental method may be applied to any turfgrass species, but more preferentially to the zoysiagrass.
The present invention also relates to the media compositions and optimal combinations of different plant growth hormones for efficient callus induction and growth. Callus induction rate was the highest when the seeds were cultured on the Murashige and Skoog (MS) medium containing 1.5-2.5 mg/L 2,4-dichlorophenoxyacetic acid, 0.15-0.25 mg/L 6-benzylaminopurine, 3.5-4.5 mg/L thiamine-HCl, and 80-120 mg/L (xcex1-ketoglutaric acid. On the contrary, callus growth was most efficient when calli was cultured on the MS medium containing 0.4-0.6 mg/L 2,4-dichlorophenoxyacetic acid, 0.015-0.025 mg/L 6-benzylaminopurine, 3.5-4.5 mg/L thiamine-HCl, and 80-120 mg/L xcex1-ketoglutaric acid.
Also, provided in the invention includes an efficient plant regeneration system. The regeneration rate was the highest when calli were transferred onto the MS medium supplemented with 2.5-3.5% maltose as a sugar source and 0.8-1.2 mg/L 6-benzylaminopurine or 0.8-1.2 mg/L thidiazuron (TDZ).
The present invention relates to the critical factors that influence the callus induction efficiency and the morphological types of calli that exhibit a high potential for shoot regeneration and a minimized frequency of albino plants. The relative ratio of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine greatly influenced the callus types. Four morphologically distinct callus types (types I-IV) were induced by different concentration ratios of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Types I-III calli readily produced shoots upon subculture. However, the watery type IV calli produced roots but not shoots. Among the types I-III calli, the type I calli exhibited the highest frequency (82%) of shoot regeneration and the lowest frequency (4%) of albinism.
Therefore, the present invention provides the media compositions and combination ratios of different growth hormones optimized for efficient induction of calli that are readily regenerated to shoots; the morphological clues that can be used to select potential calli for plant regeneration; the media components, such as sugars and cytokinins, for optimal callus induction and growth.